PROJECT SUMMARY Clusterin (CLU), also known as apolipoprotein J (ApoJ), has recently been highlighted as the third most potent genetic risk factor for late-onset Alzheimer's disease (LOAD); yet little is known about the underlying mechanisms. Our recent studies demonstrate that CLU is predominantly expressed in the brain and exists as both the mature isoform of CLU (mCLU; 75-80 kDa) and the nuclear isoform of CLU (nCLU; ~49 kDa); of particular significance, our studies reveal, for the first time, the localization of nCLU in brain mitochondria. Furthermore, our data indicates a rapid redistribution of these neuronal CLU isoforms in response to acute neurotoxic insult, including a significant removal of mCLU from the cytosol and movement of nCLU from mitochondria to cytosol of neurons. These initial findings lead to our central hypotheses that: 1) contrary to the long-held notion of nCLU as a stress-inducible and pro- apoptotic CLU isoform, nCLU may play an important role in the regulation of neuronal functions; in particular, in sustaining the mitochondrial integrity of neurons; 2) neurodegenerative stress causes the loss of mitochondrial nCLU which impairs mitochondrial stability leading to neuronal death. This project is proposed to test these hypotheses in two specific aims. In Specific Aim 1, we will first conduct a global proteomics study to identify CLU interacting partners within brain mitochondria. The CLU-mitoprotein interactions will then be examined in 3xTg-AD mice to determine how they are involved in early development of AD pathology. In Specific Aim 2, we will carry out investigations in primary neurons and brain mitochondria derived from CLU-deficient (CLU?/?) mice to examine the impact of CLU?/?, alone or in combination with cellular stress or aging, on mitochondrial morphology, movement, and bioenergetic activity. Moverover, in light of our recent finding implicating that CLU- mediated effect in the brain could be sex-specific, we will conduct all the studies proposed in this application in both female and male brains in an effort to elucidate potential sex differences in the role of CLU in brain mitochondria. Results of these first-of-its-kind exploratory studies will significantly bridge the current gap in understanding the basic properties of CLU in the brain, and provide a foundation for future in-depth studies of molecular mechanisms underlying CLU polymorphisms- conferred risk in the development of LOAD and devising strategies to mitigate the risk.